| Chinese shrimp, Fenneropenaeus chinensis, is an important breed in marine product of our country. It is delicious, and it can provide high quality proteins. So it is very popular with all consumers. However, the rapidly expanding marine industry has caused many problems. Since 1990's diseases of Chinese shrimp in breed aquatics have broken out and it has caused enormous loss in aquaculture. Many studies have been done and got success. But the immunology mechanism of shrimp is not well understood, as a difficult problem of the shrimp diseases still puzzle us.At present, people prevent and cure shrimp diseases by many methods, such as breeding bacterin, immunity intensifier and so on. The components of the immunity intensifier mainly relate to chemical matters, bacterium components, amylose, extracts of animals or plants, nutrition factors, cytokinin and so on. However, to solve the shrimp disease further we must enhance the immunity and disease resistance of the shirimp besides improving breeding environment, feed additive and the medication. So we should further study the pattern recognition acceptor of the shrimp, and we also study the mutual action among the immune factors or immune proteins in humoral immunity. In our laboratory, the immune related studies of Chinese shrimp have always been done. To research the immune related genes and their mechanism, this dissertation starts with Chinese shrimp antibacterial peptides named anti-lipopolysaccharide factor, from gene cloning to protein expression and purifaction. Our results are as follows:1. Cloning of the Chinese shrimp anti-lipopolysaccharide factor gene (ALF) .According to the conservative sequence of ALF gene from other close species, we designed the degenerated primers to clone the mid fragment of Chinese shrimp's ALF gene. After many times experiments, we found the optimum condition and successfully gained the mid fragment of the cDNA about 280 bp.To clone the 3' and 5' ends of ALF gene the sequence of the mid fragment was used for design the primers. With these primers joining as partners of 3' anchor R and 5' PCR primer, the 3' and 5' end of the cDNA had been cloned. We combine the fragments together and get the whole-length ALF cDNA of Chinese shrimp.2. Construction of the recombinant plasmidThis dissertation indicated the process of structuring the recombinant plasmid, pET-30a(+)-ALF and pGEX 4T-1-ALF.3. Expressing anti-lipopolysaccharide factor in Escherich coli.We transformated the recombinant carrier, pGEX 4T-1-ALF to E.coli BL21, and studied the expression of anti-lipopolysaccharide factor gene in prokaryocyte, and purified the products of ALF gene. During the process of expression we successfully resolved the problem of the low expression with the rare codons in E.coli. This practice can furnish a new base in the theory and practice for increasing the expressin by changing rare codons.Medically LPS relates to systemic inflammation response syndrome, endotoxic shock, sepsis and matiple organ dysfunction syndrome, and it threatens human's health. Anti-lipopolysaccharide factor can integrate the LPS's toxicity, so it can be used to treat and prevent diseases mentioned above. Besides, Lipoposaccharide is the main component of the outer membrane of gram-nagtive's cell wall, so ALF has antibacterial activity. As an antibacterial peptide has a wide application prospect. In my dissertation the study is mainly about cloning and expression of ALF gene. It can provide a theoretic basis for prevention and treatment of prawn disease. However, some shortcomings still remain in my experiments, such as to study the recombinant protein's antibacterial activity, and it's fuction of resistant disease of human being.
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